The mitochondrial genomes of all organisms encode only a small fraction of the gene products necessary for mitochondrial biogenesis and function, with the remainder being encoded in the nucleus. This arrangement requires that the expression of the nuclear genes be coordinated with that of the mitochondrial genes. Abnormalities in mitochondrial expression or function are the underlying cause of a heterogeneous group of human diseases termed the mitochondrial myopathies. At least two of these myopathies are caused by nuclear mutations; some may be caused by a breakdown in communication between nucleus and mitochondrion. We have shown that in Saccharomyces cerevisiae, a mitochondrial DNA segment (containing a GC cluster belonging to the M3 class) that is located adjacent to an in vivo transcription initiation site has the capacity to act as a mitochondrial promoter in vitro, and has the surprising capacity to at as a catabolite-repressible nuclear promoter in vivo. We have also shown that several factors bind to the GC cluster, that the presence of one of the binding activities. (MBF1, found both inside and outside of the mitochondria) is catabolite- repressible, i.e., binding is strictly correlated with the appearance of both GC cluster-dependent nuclear transcription and active mitochondria. Another link between MBF1 binding activity and GC cluster-dependent nuclear transcription is that they are both depressed in a hex2 mutant. Although no naturally occurring GC clusters have been found in the nuclear DNA, one or more nuclear factors that bind to them appear to play an important role, since titration of the GC cluster binding factors in the nucleus in vivo drastically reduces the capacity of yeast cells to grow on a non- fermentable carbon source. These data suggest that one or more of the binding activities is involved in the coordination of nuclear and mitochondrial expression. In this proposal, I describe plans to (1) generate mbf mutants, (2) biochemically purify MBF1, (3) use the purified MBF1 to generate polyclonal antisera and derive a partial peptide sequence, (4) use the antibody to test the involvement of MBF1 in the in vitro transcription assay, and determine directly the cellular localization of MBF1 during aerobic and anaerobic growth, (5) clone the gene(s) encoding this factor either by using the antibody, oligonucleotides or complementation of the mbf mutants, (6) transform mitochondria to assess the role of the M3 GC cluster in the mitochondrial genome, and (7) begin a functional analysis of MBF1 (by gene disruption and in vitro mutagenesis) to determine its role(s) in the nucleus and the mitochondria. The successful completion of this project will contribute not only to our knowledge of nuclear transcriptional regulation and mitochondrial biogenesis, but also the means by which eukaryotic cells coordinate both processes. Ultimately, this knowledge will be useful in understanding and eventually treating some of the devastating human diseases caused by mitochondrial dysfunction.